An Enzyme-Linked Immunosorbent Assay for the Detection of Mitochondrial DNA–Protein Cross-Links from Mammalian Cells

DNA–Protein cross-links (DPCs) are cytotoxic DNA lesions with a protein covalently bound to the DNA. Although much has been learned about formation, repair, and biological consequences of DPCs in nucleus, little is known regarding mitochondrial DPCs. This due part lack robust specific methods measure Herein, we reported an enzyme-linked immunosorbent assay (ELISA)-based method for detecting formed between transcription factor A (TFAM) cultured human cells. To optimize purification detection workflow, prepared model TFAM-DPCs via Schiff base chemistry using recombinant TFAM substrate containing abasic (AP) lesion. We optimized isolation commercial silica gel-based columns achieve high recovery yield evaluated microplate, DNA-coating solution, HRP sensitive TFAM-DPCs. Additionally, mtDNA procedure eliminate almost all nuclear contaminants. For proof concept, detected different levels from HEK293 cells under conditions. The based on commercially available materials can be amended detect other types mitochondria.